Microfluidic platform for multiplexed detection in single cells and methods thereof

Abstract

The present invention relates to a microfluidic device and platform configured to conduct multiplexed analysis within the device. In particular, the device allows multiple targets to be detected on a single-cell level. Also provided are methods of performing multiplexed analyses to detect one or more target nucleic acids, proteins, and post-translational modifications.

The invention claimed is: 
     
       1. A method for performing multiplexed, single-cell analysis in a microfluidic device, the method comprising:
 (i) loading a cell sample into a device, wherein the device comprises a plurality of holding chambers; 
 (ii) holding a portion of the cell sample within each holding chamber, thereby providing a held cell; 
 (iii) incubating the held cell with a first protein label in each holding chamber, wherein the first protein label is configured to detect a first target protein, thereby providing a labeled cell; 
 (iv) incubating the labeled cell with a first nucleic acid label, wherein the first nucleic acid label is configured to detect a first target nucleic acid, thereby providing a multiplexed-labeled cell; 
 (v) detaching the multiplexed-labeled cell, thereby providing a detached, intact cell; and 
 (vi) performing an on-chip flow cytometry assay of the detached, intact cell, thereby performing multiplexed, single-cell analysis for the target protein and/or the target nucleic acid. 
 
     
     
       2. The method of  claim 1 , further comprising:
 stimulating the cell sample with a stimulant. 
 
     
     
       3. The method of  claim 1 , wherein the first protein label and/or the first nucleic acid label further comprises a detectable marker. 
     
     
       4. The method of  claim 1 , wherein the first nucleic acid label further comprises a first affinity agent, and the method further comprises, after step (iv), incubating the multiplexed-labeled cell with a secondary label comprising a second affinity agent, wherein the first and second affinity agents are configured to bind together. 
     
     
       5. The method of  claim 4 , wherein the method further comprises incubating the multiplexed-labeled cell with one or more tertiary labels, wherein each tertiary label is, independently, configured to bind to the secondary label and independently comprises a proximity ligation probe; and incubating with one or more quaternary labels, wherein each quaternary label is, independently, configured to bind to a portion of the proximity ligation probe. 
     
     
       6. The method of  claim 1 , further comprising:
 amplifying a signal of the first nucleic acid label. 
 
     
     
       7. The method of  claim 6 , wherein the amplifying step comprises performing a rolling circle amplification by providing one or more affinity agents configured to bind to the first nucleic acid label, one or more proximity ligation probes configured to bind at least one affinity agent, one or more connector probes configured to bind at least one proximity ligation probe and to form a circular template, and one or more enzymes configured to generate a concatemer based on the circular template. 
     
     
       8. The method of  claim 1 , further comprising:
 incubating the labeled cell with a second protein label in each assay chamber, wherein the second protein label is configured to detect a second target protein. 
 
     
     
       9. The method of  claim 8 , wherein the first target protein is a cell surface protein and the second target protein is an intracellular protein. 
     
     
       10. The method of  claim 1 , wherein step (vi) comprises hydrodynamically focusing the detached, intact cell to a center of the device. 
     
     
       11. The method of  claim 1 , further comprising:
 incubating the held cell with a post-translation modification label or a glycosylation label. 
 
     
     
       12. The method of  claim 1 , wherein the plurality of holding chambers comprises a plurality of individually addressable holding chambers. 
     
     
       13. The method of  claim 12 , wherein each of the plurality of individually addressable holding chambers is connected to a programmable valve and a pump. 
     
     
       14. The method of  claim 1 , wherein the first nucleic acid label is a miRNA label or a mRNA label. 
     
     
       15. The method of  claim 1 , wherein the first protein label is a cytosolic protein label, a cell surface receptor label, a cell surface protein label, a post-translational modification label, a dynamic glycosylation label, an intracellular protein label, or a phospho-protein label. 
     
     
       16. The method of  claim 1 , wherein step (iv) comprises conducting a fluorescent in situ hybridization assay. 
     
     
       17. The method of  claim 16 , wherein the fluorescent in situ hybridization assay comprises locked nucleic acid probes.